Abbreviations: CMS protein, connective membrane skeleton protein; FA, focal adhesion; NLS, nuclear localization signal; NES, nuclear export signal. NM, nuclear matrix; rBM, reconstituted basement membrane; HMEC, human mammary epithelial cells; 2D and 3D, two and three dimensional; Rb, retinoblastoma protein; ECM, extracellular matrix; EGF, epidermal growth factor; mAb, monoclonal antibody;
The cell nucleus is organized by a non-chromatin internal structure referred to as the nuclear matrix (NM). Identified NM components include coiled-coil proteins, cell cycle regulators, tissue-specific transcription factors, and RNA splicing factors. Although splicing factors have been shown to redistribute during cellular differentiation, and following the induction of gene expression, such alterations in nuclear organization, defined here as the spatial distribution of nuclear components, are thought to be the consequence of changes in gene expression. However, both NM composition and structure may affect gene expression and cellular function, and thus a systematic analysis of nuclear organization during such a complex process as tissue differentiation is warranted.
To study the effect of cell growth and tissue differentiation on nuclear organization, we have used a reconstituted basement membrane (rBM)-directed model of mammary gland morphogenesis. The HMT-3522 human mammary epithelial cells (HMECs) were isolated from reduction mammoplasty and became immortalized in culture. When embedded within a rBM, these cells growth arrest, organize an endogenous BM and form polarized acinus-like structures with vectorial secretion of sialomucin into a central lumen. Using this model, we have compared the nuclear organization of HMECs cultured on a plastic surface (2D monolayer) vs. a 3-dimensional (3D) rBM. Nuclear organization was assessed by examining the distribution of the coiled-coil NM proteins lamin B and NuMA, the cell cycle regulator Rb (p110Rb; 5), and the splicing factor SRm 160 (formerly known as B 1 C8; 16). These proteins had distinct spatial distribution patterns specific for proliferation, growth-arrest and acini formation. Moreover, disruption of nuclear organization in acini by either perturbing histone acetylation, or directly modifying the distribution of NuMA proteins, altered the acinar phenotype.
We have previously determined that the extracellular matrix (ECM) directs morphogenesis and gene expression in mammary epithelial cells. Here we show that a reciprocal relationship exists between the ECM and nuclear organization. These findings underscore a role for nuclear organization in regulation of gene expression and provide a possible framework for how cell-ECM interactions determine cell and tissue phenotype.